| Lung cancer is one of the most malignant tumors both in China and worldwide with poor morbidity and mortality.About 85% of lung cancers are non-small cell lung cancer(NSCLC),which mainly including squamous cell lung cancer and adenocarcinoma.Becaouse of the difficulty of early diagnosis and drug resistance,the 5-year survival rate is less than 20%.Therefore,it is important to find new diagnostic markers.As a hotspot in recent years,lncRNAs have important roles in lung cancer.Long non-codingRNA(lncRNA)refers to the non-codingRNA with a length of more than 200 nucleotides.LncRNAs can participate in the regulation of various cellular processes.And it can participate in the regulation of many biological processes,such as intracellular X chromosome silencing,gene imprinting,chromatin remodeling,transcriptional activation,transcriptional interference,intranuclear transport and so on.In order to reveal the mechanism of key lncRNA molecules in NSCLC,we detected the expression of differential molecules in 8 pairs of NSCLC tissues and their matched normal lung tissues by Arraystar Human lncRNA/mRNA.We found that 4 lncRNAs,were highly expressed in cancer tissues,including FEZF1-AS1,Linc01296,DUXAP8 and DUXAP10.This study will focus on the expression,functions and mechanism of FEZF1-AS1 in non-small cell lung cancer,including three sections: 1.The expression of FEZF1-AS1 in non-small cell lung cancer and its functions in vitro;2.FEZF1-AS1 regulates the expression of miRNAs in non-small cell lung cancer as ceRNA;3.The role of FEZF1-AS1 in regulating the expression of miR-516b-5p-ITGA11 in non-small cell lung cancer.It is hoped that the above studies will clarify the role of FEZF1-AS1 in non-small cell lung cancer and provide a new marker for non-small cell lung cancer.Section? The expression of FEZF1-AS1 in non-small cell lung cancer and its functions in vitro.Objective: To verify the high expression of FEZF1-AS1 in non-small cell lung cancer(NSCLC),and to observe the effect of FEZF1-AS1 on NSCLC cells in terms of cell proliferation,migration,apoptosis and cell cycle distribution,and to explore the factors affecting the expression of FEZF1-AS1.Methods: The expression of FEZF1-AS1 was detected by qRT-PCR in45 pairs of NSCLC specimens and their corresponding normal lung tissues,and the relationship between FEZF1-AS1 expression and clinicopathological features was analyzed.FEZF1-AS1 was knocked down by siRNA transfection in H1299 and H520 cells,then the cell proliferation was detected by CCK8,cell migration and invasive ability were detected by Transwell chamber test,cell cycle and apoptosis were detected by flow cytometry.The experiment was repeated three times in each group.All data were analyzed by SPSS Statistics23.0,P<0.05 means significant difference.Results:1 FEZF1-AS1 is highly expressed in non-small cell lung cancer and is associated with lymph node metastasis.We analyzed the expression of lncRNA/RNA in 8 pairs of non-small cell lung cancer tissues(4 pairs of squamous cell carcinomas tissues and 4 pairs of adenocarcinomas tissues)and their matched normal lung tissues by Arraystar Human lncRNA/RNA microarray technology.Under FC?2,P<0.05 condition,505 lncRNAs were significantly up-regulated and 652 lncRNAs were significantly down-regulated.Further more,20 up-regulated lncRNAs and 26down-regulated lncRNAs were selected with FC?5,P<0.05 as the limiting conditions,and 15 lncRNAs were selected from them.The results that further verified in 20 pairs of lung cancer tissues and matched normal lung tissues showed that the expressions of FEZF1-AS1,DUXAP8,Linc01296 and DUXAP10 in cancer tissues were significantly higher than those in normal tissues.This study only focused on FEZF1-AS1.Quantitative RT-PCR was used to detect the expression of FEZF1-AS1 in 45 pairs of NSCLC specimens and corresponding normal lung tissues.It was found that the expression of FEZF1-AS1 in cancer tissues was significantly higher than that in normal tissues(P<0.0001).FEZF1-AS1 was divided into high expression group(n=30)and low expression group(n=15)by rule of thirds.The relationship between the expression of FEZF1-AS1 and clinicopathological features was analyzed.It was found that the high expression of FEZF1-AS1 had significant lymph node metastasis(P=0.020).Through drawing AUC curve,we can see that FEZF1-AS1 had good diagnostic significance for NSCLC(AUC = 0.759,P< 0.0001).2 Expression and function of FEZF1-AS1 in non-small cell lung cancer cell linesFEZF1-AS1 is highly expressed in non-small cell lung cancer cell lines.CCK8 assay showed that the cell proliferation was significantly inhibited after knockdown FEZF1-AS1(P<0.05).Compared with NC group,the number of perforating cells in si-FEZF1-AS1 group significantly decreased in H1299(P<0.05)and H520(P<0.05).In invasion experiment,the number of si-FEZF1-AS1 cells decreased compared with NC cells(P<0.05).Flow cytometry was used to detect the effect of knockdown FEZF1-AS1 on cell cycle and apoptosis.It was found that knockdown FEAF1-AS1 resulted in G2/M phase arrest in NSCLC cells(P<0.05),but had no significant effect on cell apoptosis.3 m~6A modification regulates the expression of FEZF1-AS1The full-length sequence of FEZF1-AS(2,653 bp)was predicted by m~6A prediction software SRAMP.Seven m~6A modification sites were predicted,suggesting that the expression of FEZF1-AS1 may be regulated by m~6A modification.Knockdown YTHDF1,YTHDF2,METTL3 and METTL14 could down-regulate the expression of FEZF1-AS1 respectively.The expression of FEZF1-AS1 in H1299 and H520 treated with 3-Deazaadenosine at a concentration of 10?M was detected in three groups: untreated,4 hours treated and 24 hours treated.Compared with untreated group,the expression of FEZF1-AS1 was significantly decreased at 4-hours,and it was increased at24 hours compared with untreated group.Summary:1.FEZF1-AS1 is highly expressed in non-small cell lung cancer,and its high expression is significantly related to lymph node metastasis,which can be used as a biomarker of diagnosis and prognosis.2.Knockdown the expression of FEZF1-AS1 can significantly inhibit the proliferation,cell cycle progression,migration and invasion of NSCLC cells,suggesting that FEZF1-AS1 may play an oncogene role in NSCLC.3.m~6A modification can significantly regulate the expression of FEZF1-AS1 in NSCLC cells.Section? FEZF1-AS1 regulates the expression of miR-320e/940-mRNAs in non-small cell lung cancer as ceRNAObjective: From the perspective of the role of ceRNA,the possible mechanism of FEZF1-AS1 oncogene in NSCLC was discussed.Methods: Probable miRNAs were screened out by ceRNA prediction analysis,and then the miRNAs mimics library was constructed and transfected into cells.The changes of FEZF1-AS1 expression were detected,and miRNAs with the most significant decrease in expression were selected as miR-320 e and miR-940.MiR-320 e and miR-940 were overexpressed in H1299 and H520 cells.CCK8 assay was used to detect cell proliferation,Transwell chamber assay was used to detect cell migration and invasion,and flow cytometry was used to detect cell cycle.The target genes of miR-320 e and miR-940 were respectively identified as E2F1 and SRC.Their relationships with miR-320 e,miR-940 and FEZF1-AS1 were verified by PCR and Western blot.The binding of FEZF1-AS1 with miR-320 e and miR-940 was verified by RIP and RNA pull-down experiments.The experiment was repeated three times in each group.All data were analyzed by SPSS Statistics 23.0,P<0.05 means significant difference.Results:1.FEZF1-AS1 mainly distributes in the cytoplasm and regulates withmiRNA-320 e and miRNA-940.Nuclear-cytoplasmic separation experiments showed that FEZF1-AS1 was mainly distributed in the cytoplasm.Twelve microRNAs were screened by ceRNA prediction and analysis,and then made into a miRNAs mimics library.The expression of FEZF1-AS1 was detected after over-expression of miRNAs.It was found that the expression of FEZF1-AS1 was decreased most significantly by miR-320 e and miR-940.2.Expression of miR-320 e and miR-940 in non-small cell lung cancerThe expression of miR-320 e and miR-940 in 397 NSCLC tissues and 151 normal lung tissues were retrieved by GEO database(GSE 53882).The results showed that both miR-320e(P<0.05)and miR-940(P<0.001)were significantly lower in cancer tissues.In 45 cases of NSCLC and its matched lung tissues,the expression of miR-320 e was significantly lower in 55.56%(25/45)of the tumors,while the expression of miR-940 was significantly lower in 31.11%(14/45)of the tumors.3.Functions of miR-320 e and miR-940 in non-small cell lung cancer cell linesmiR-320 e mimics and miR-940 mimics were transfected into H1299 cells,while NC mimics was the control group,and the expression of them increased significantly at the later stage of transfection(P<0.05).miR-320 e mimics were transfected into H1299 and H520 cells respectively,and NC mimics were used as control group.CCK8 assay showed no significant difference in cell proliferation.Transwell assay showed that the number of miR-320 e over-expression group was significantly lower than that of NC group(P<0.05).Mi R-940 mimics were transfected into H1299 and H520 cells respectively,and NC mimics were used as control group.CCK8 assay showed significant difference in cell proliferation.Transwell chamber assay showed that the number of miR-940 overexpression group was significantly lower than that of NC group(P<0.05).4.FEZF1-AS1 participates in the regulation of miR-320e-E2F1 as ceRNAThe downstream target genes regulated by miR-320 e were screened and validated,including E2F1 and SKA3 genes.WB results confirmed that over-expression of miR-320 e could significantly inhibit the expression of E2F1 protein.At the same time,knockdown the expression of FEZF1-AS1 in H1299 and H520 cells can reduce the expression of E2F1 both in mRNA level and protein level.RNA pull-down assay showed that FEZF1-AS1 was significantly enriched in the bio-miR-320e-WT transfection group(P<0.05)compared with the negative control bio-miR-NC,while the expression of FEZF1-AS1 was not detected in the bio-miR-320e-MUT group.On the basis of the above detection,we further observed the role of FEZF1-AS1 in the regulation of E2F1 by miR-320 e through response experiments.The results showed that overexpression of FEZF1-AS1 could reverse the inhibitory effect of miR-320 e on E2F1 to some extent.The results showed that FEZF1-AS1 as ceRNA participated in the regulation of E2F1 by miR-320 e in NSCLC.5.FEZF1-AS1 participates in the regulation of SRC by miR-940 as ceRNAThe downstream target genes regulated by miR-940 were screened and validated,including SRC and SKA3 genes.WB results confirmed that over-expression of miR-940 could significantly inhibit the expression of SRC protein.At the same time,knockdown the expression of FEZF1-AS1 in H1299 and H520 cells can reduce the expression of SRC at the level of mRNA and protein.RNA pull-down assay showed that FEZF1-AS1 was significantly enriched in the Bio-miR-940-WT transfection group compared with the negative control Bio-miR-NC(P<0.05),while the expression of FEZF1-AS1 was not detected in the Bio-miR-940-MUT group.On the basis of the above detection,we further observed the regulatory effect of FEZF1-AS1 on SRC by reply experiment.The results showed that overexpression of FEZF1-AS1 could reverse the inhibitory effect of miR-940 on SRC to a certain extent.The results showed that FEZF1-AS1 as ceRNA participated in the regulation of SRC by miR-940 in NSCLC.Summary:1.FEZF1-AS1 mainly distributes in the cytoplasm and binds to Ago2 protein,suggesting that there is a mechanism of ceRNA interaction between FEZF1-AS1 and miRNAs.2.Overexpression of miR-320 e and miR-940 can significantly inhibit the migration,invasion and proliferation of NSCLC cells,suggesting that they have anti-oncogene effect in NSCLC.3.FEZF1-AS1,as the regulation of ceRNA on miR-320e-E2F1 and miR-940-SRC,is an important mechanism of its involvement in the genesis and development of NSCLC.Section? The role of FEZF1-AS1 in regulating the expression of miR-516b-5p-ITGA11 in non-small cell lung cancerObjective: To investigate the regulation and mechanism of miR-516b-5p-FEZF1-AS1-ITGA11 in non-small cell lung cancer.Methods: Possible mRNAs were screened out by microarray results,FEZF1-AS1 was knocked down in cells,and the changes of targeted genes were detected.ITGA11 with the lowest expression was selected and verified by western blot.After the transfection efficiency was verified by PCR and western blot in H1299 and H520 cells respectively,ITGA11 was knocked down,then the cell proliferation was detected by CCK8 method,the cell migration and invasive ability were detected by Transwell chamber test,and the cell cycle was detected by flow cytometry.Results:1.FEZF1-AS1 regulates the expression of ITGA11Predictive of ceRNA and GO analysis were performed,and the results were cross-matched with those of non-small cell lung cancer in the early stage of this study.Nine possible target genes,ITGA11,RHOV,ONECUT2,SOX4,MARK4,UBN1,COL1A1,PRKAB2 and PIP5K1 A,were selected.After knockdown FEZF1-AS1 in H1299 and H358 cells,the expression of these nine genes was detected,and ITGA11 was the lowest in both two cells.To further verify,si-FEZF1-AS1 and negative control si-NC were transfected into H1299 and H520 cells respectively.The expression level of ITGA11 protein was detected,which showed that the expression of ITGA11 decreased in knockdown group.2.Expression of ITGA11 in NSCLC and its biological roleThe expression of ITGA11 in 45 tumor tissues was significantly higher than that in normal lung tissues(P<0.05).ITGA11 was transfected into NSCLC cells,and the transfection efficiency was confirmed byRNA and protein.CCK8 test showed that the cell proliferation was significantly inhibited after knocking down ITGA11(P<0.05).In migration experiment,the number of perforating cells in knockdown group decreased significantly in H1299(P<0.05)and H520(P<0.05).In invasion experiment,the number of si-ITGA11 cells in H1299 cells was less than that in NC cells(P<0.05),but there was no significant change in H520 cells.Flow cytometry showed that knockdown FEZF1-AS1 had no significant effect on cell cycle.3.Prediction and screening of ITGA11-related microRNAsThe miRNAs targeting ITGA11 were screened and compared with the results of FEZF1-AS1 ceRNA prediction analysis and GO analysis.From then on,six miRNAs were selected,miR-126a-3p,miR-29 b,has-miR-145,miR-486-3p,miR-516b-5p and miR-584-3p.The expression changes of ITGA11 and FEZF1-AS1 were detected after transfection of the six miRNA mimics.It was found that the miR-516b-5p had the strongest effect in two cells.The expression of ITGA11 was detected after miR-516 b mimics transfection.The expression of ITGA11 was decreased in the miR-516 b mimics group.4.Expression and biological role of miR-516b-5p in non-small cell lung cancerThe expression of miR-516b-5p was significantly lower than that in normal lung tissues in 45 pairs of tissues(P<0.01).The proliferation,migration and invasion of NSCLC cells were respectively inhibited by over-expression of miR-516 b.5.Binding of miR-516b-5p with FEZF1-AS1Bio-miR-516b-MUT,Bio-miR-516b-WT and negative control Bio-miR-NC were transfected with biotin markers.RNA pull-down assay was used to detect the enrichment of FEZF1-AS1.It was found that FEZF1-AS1 was significantly enriched in WT group(P<0.05).Combining with the decrease of FEZF1-AS1 expression after the overexpression of the former miR-516 b,we can conclude that miR-516b-5p and FEZF1-AS1 have a binding effect and compete with the downstream gene ITGA11.6.FEZF1-AS1-miR-516b-5p-ITGA11 recovery experimentNC,miRNA-516 b mimics and pc DNA3.1 FEZF1-AS1+miR-516 b mimics were transfected respectively.After 48 hours,the protein was extracted to detect the expression of ITGA11.Compared with NC group,the ITGA11 expression of miR-516 b mimics group was lower,and the co-transfection group was higher than that of miR-516 b mimics group.Summary:1.The expression of ITGA11 in NSCLC tumors was significantly higher than that in matched normal lung tissues.Knockdown ITGA11 could significantly inhibit the proliferation,migration and invasion of NSCLC cells,suggesting that ITGA11 acts as oncogene in NSCLC.2.The expression of miR-516b-5p in NSCLC tumors was significantly lower than that in matched normal lung tissues.Overexpression of miR-516b-5p could inhibit cell proliferation,migration and invasion,suggesting that miR-516b-5p plays an important role in NSCLC.3.FEZF1-AS1 participates in the development of NSCLC by regulating miR-516b-5p-ITGA11 pathway as a ceRNA.Conclusions:1.FEZF1-AS1 is highly expressed in non-small cell lung cancer,and its expression is positively correlated with lymph node metastasis.Knockdown FEZF1-AS1 can significantly inhibit the proliferation,cell cycle progression,migration and invasion of NSCLC cells.Combined with AUC curve analysis,FEZF1-AS1 has oncogene functions in NSCLC and can be used as a biomarker for diagnosis and prognosis of NSCLC.2.FEZF1-AS1 mainly distributes in the cytoplasm and binds to Ago2 protein,suggesting that FEZF1-AS1 may participate in the development of NSCLC as ceRNA.3.Overexpression of miR-320 e,miR-940 and miR-516b-5p can significantly inhibit the migration,invasion and proliferation abilities of NSCLC cells,suggesting that the above-mentioned miRNAs act as anti-oncogene in NSCLC.4.FEZF1-AS1 participates in the genesis and development of NSCLC by regulating miR-320e-E2F1,miR-940-SRC and miR-516b-5p-ITGA11 pathways as ceRNA.5.m~6A modification is involved in the expression of FEZF1-AS1 in NSCLC cells. |
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